Since the pioneering work of Updike and Hicks, Nature (London), Vol. 214 (1971) 986, there has been much effort devoted to creating bioanalytical measurement devices by immobilizing an enzyme in, on, or around a measurement device thus creating a biosensor. These so called biosensors find wide application to the field of analytical, environmental and clinical chemistry as well as in the practice of medicine. The principal function of the enzyme(s) in a biosensor is to catalyze the conversion, with a high degree of specificity, of a substance, whose concentration or presence is being sought, into a species that may be readily detected with a chemically selective sensor.
One example of such an application is the combination of urease, an enzyme that specifically hydrolyzes urea to ammonium ions, with an ion-selective electrode or sensor that selectively responds to ammonium ions. Such sensor could have a membrane containing the ionophore nonactin. An ammonium selective sensor would not, in the absence of the enzyme, be able to detect or measure urea.
Another widely used biosensor provides the direct measurement of glucose in physiological fluids. Most common glucose biosensors depend on glucose oxidase catalyzing the oxidation of glucose to hydrogen peroxide. In turn, the concentration of hydrogen peroxide is measured using an amperometric electrochemical cell. The magnitude of the current flowing in the cell is linearly related to the concentration of glucose, as described in U.S. Pat. No. 4,759,828 which is hereby incorporated by reference.
Although many articles related to sensor design, fabrication and use can be found in the technic, al and patent literature, it is widely acknowledged that, to date, there have been very few truly practical biosensors. The technical challenges are many. A notable challenge is maintaining the level of biological activity of the enzyme(s) used in the biosensor so that it may be used for multiple applications.
One approach to achieving and maintaining a high level of enzyme activity, involves mixing the sample of interest with a solution/preparation of the enzyme, immediately before and during the time of measurement. Although this approach does not suffer from the complexities of creating a stable, active, immobilized enzyme, it is fraught with the problem of providing a means to continually add the enzyme containing solution. This increases the complexity of an apparatus used for such analyses and significantly more enzyme is consumed compared with the ideal of a stable, active immobilized enzyme in, on or around the measuring sensor.
Techniques of achieving immobilization of an active enzyme are documented in the technical literature. Enzymes are commonly either adsorbed or covalently attached to, or in close proximity to, the measuring sensor or incorporated into a membrane that is positioned between the sample and the measuring device. Conditions are established to allow the product(s) of the enzymatic process to either interact with a selective sensor surface (an ionophoric layer in the case of a potentiometric device) or diffuse through the membrane to the working electrodes (in the case of an amperometric device). Unfortunately, many biosensors based on such approaches often show disappointing performance. Typical problems include slow hydration of the membrane or degradation of the enzyme by components in the sample or calibration fluids (heavy metals, bacteria or proteases--enzymes that destroy proteins).
In clinical applications, membrane based enzyme systems are preferred since samples can, and do, contain numerous unspecified components that can harm the enzyme used in the biosensor. The membrane matrix is selected to be porous to the test substance, any cofactors or reagents needed by the enzyme, and the byproducts of the reaction. Ideally the membrane structure provides an enzyme-friendly environment while acting as a barrier to agents that will harm the enzyme.
Commercial systems that use biosensors are relatively complex. An advanced system design enables a series of calibration fluids or reagents to be passed sequentially over the biosensors in order to maintain enzyme activity and to calibrate the devices. Good analytical performance is achieved only by rigorous maintenance of the protocol or fluid sequence used. This virtually dictates the need for a computer controlled system.
Notwithstanding the aforementioned efforts, it is apparent that enzyme based sensors do not last indefinitely. Since enzymes represent a significant part of the overall cost of reagents, it is desirable to maximize their useful life. Single use sensors are typically cost prohibitive except in the most specialized applications. Moreover, single use devices, by definition, cannot be calibrated with an authentic fluid to verify functionality and then be used to test a sample.
Multiple use of sensors is cost effective provided protocols for removing prior sample components and reaction byproducts are effective. One method employed by commercial manufacturers is to sequence relatively large volumes of flushing and/or calibration fluids past the sensors. To be effective, refreshing and/or calibrating the sensors is necessary throughout the entire life of the system, whether or not testing is in progress. For any reasonable period of unattended use, it follows that the system must accomodate relatively large volumes of fluid reagents. While this is of little concern for analyzers designed for laboratory use, the problem is more acute for portable or hand held analyzers.
An alternative fairly effective method of extending sensor life is to reduce the volume of sample either by reducing the scale of operation or by pre-diluting the sample. The objective in either case is to limit the amount of substrate a given amount of enzyme the sensor must process and to reduce the overall amount of byproducts. Limiting the sample size with or without dilution tends to increase sensor life and some economy of fluids is achieved. However, attempts to reduce the extent of flushing or calibration invariably leads to a shorter sensor life.
Few manufacturers offer systems based on multiple bioanalytical systems capable of sensing multiple analytes. Even here, sensor life problems appear to be limiting this practical utilization of sensors. Notable exceptions are i-STAT, (Princeton, N.J.) who solves the sensor lifetime problem by employing a single use multianalyte disposable device. One of their systems is described in U.S. Pat. No. 5,063,081.
Another multiuse analyzer is described in U.S. Pat. No. 4,452,682 which tests blood gasses, electrolytes and metabolites. The patent describes a sequential system in which the glucose sensor is positioned upstream of the urea sensor. This system does nothing to enhance the lifetime of the sensor. These prior art sequential flow systems position a glucose sensor which generates hydrogen peroxide (H.sub.2 O.sub.2) upstream of at least some of other metabolite, i.e., biosensors. This tends to shorten the lifetime of these downstream sensors as described.
NOVA Biomedical (Waltham, Mass.) sells multianalyte sensors and relies on relatively large volumes of flush fluids to assure practical use life. Analyzers marketed by the latter manufacturer use a flow channel to sequentially flow samples and calibration fluids past electrolyte sensors, e.g., sodium, potassium and chloride and then past biosensors for creatinine, glucose and urea.